title: Residue Phe42 is critical for the catalytic activity of Escherichia coli major nitroreductase NfsA.
abstract: The major O2-insensitive nitroreductase (NfsA) of Escherichia coli shares low sequence homology but similar biochemical and structural features with NfsB, the E. coli minor O2-insensitive nitroreductase. A structural comparison revealed Phe42 was present in the active site of NfsA but not NfsB. F42Y, F42N and F42A were generated and had decreased activity toward nitrofurazone by 52, 96, and 99%, respectively. The kinetic parameters for other nitroaromatic substrates were also determined. Compared to wild type, the mutants did not have significantly altered K(m)s, but had dramatically decreased k(cat) and k(cat)/K(m) values. Far-UV CD spectral analysis of the mutants suggested that there were no significant conformational changes however F42A and F42N had changes from 208 to 222 nm, which was attributed to loss of helix content. These findings revealed that Phe42 is important for maintaining NfsA activity and structure.
authors.affiliation: School of Life Science & Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian, 116023, People's Republic of China.
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.forename: Junfei
authors.initials: J
authors.lastname: Zhan
authors.forename: Jing
authors.initials: J
authors.lastname: Bai
authors.forename: Peiyu
authors.initials: P
authors.lastname: Liu
authors.forename: Yingjiao
authors.initials: Y
authors.lastname: Xue
authors.forename: Qing
authors.initials: Q
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009601:Nitroreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment
chemical_list: D029968:Escherichia coli Proteins; D050505:Mutant Proteins; C116053:NfsA protein, E coli; D009601:Nitroreductases